Conjugated polymeric particle and method of making same

ABSTRACT

A method of conjugating a substrate includes exchanging a counter ion associated with a biomolecule with a lipophilic counter ion to form a biomolecule complex, dispersing the biomolecule complex in a nonaqueous solvent, and coupling the biomolecule complex to a substrate in the presence of the nonaqueous solvent.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application is a continuation application under 35 U.S.C. § 120 ofpending U.S. application Ser. No. 15/945,348 filed Apr. 4, 2018, whichis a divisional application of U.S. application Ser. No. 14/855,705filed Sep. 16, 2015 (now U.S. Pat. No. 9,938,577), which is acontinuation of U.S. application Ser. No. 13/763,066 filed Feb. 8, 2013(now U.S. Pat. No. 9,139,667), which application claims the benefitunder 35 U.S.C. § 119(e) of U.S. Provisional Application No. 61/597,064filed Feb. 9, 2012. The entire contents of the aforementionedapplications are incorporated by reference herein.

FIELD OF THE DISCLOSURE

This disclosure, in general, relates to methods of conjugating asubstrate and substrates formed through such methods.

BACKGROUND

The conjugation of polynucleotides to substrates has become of interestto various industries. Substrates including conjugated polynucleotidesare useful in separation techniques, detection of genetic markers, andsequencing.

For example, a substrate conjugated with a polynucleotide probe can beused for capturing genetic markers for detection. Exemplary geneticmarkers can be related to disease variants within a gene, diseasecausing bacteria or viruses, or alleles useful in human identification.Substrates including conjugated probes complementary to a genetic markercan capture such a genetic marker, and various techniques can be used todetect the presence of the captured genetic marker.

In another example, a substrate conjugated with a polynucleotide can beuseful in capturing and separating genetic material. In an example,probes on a substrate can be complementary to a desired polynucleotide.The probes can be configured to capture the desired polynucleotide andlater release the polynucleotide to permit recovery of thepolynucleotide. In another example, polymeric particles conjugated withpolynucleotide probes that are complementary to target polynucleotidescan be used to capture and separate a target polynucleotide fromsolution. Subsequently, the target polynucleotide can be released fromthe conjugated particle in a different solution.

In a further example, a substrate conjugated to a polynucleotide can beused in various sequencing techniques. For example, a polymeric particleconjugated to a polynucleotide or multiple copies of the polynucleotidecan be used in sequencing techniques, such as fluorescent-basedsequencing techniques or ion-based sequencing techniques.

While each of the above uses of conjugated substrates is of particularinterest to various industries, reliable conjugation of a polynucleotideto a substrate, such as a polymeric substrate, is often inefficient.Such inefficiencies lead to lower densities of the conjugatedpolynucleotide or random regions devoid of the desired polynucleotide.Such inefficiencies can result in poor separations, low accuracy indetection methods, and low signal or high signal-to-noise ratio insequencing techniques.

As such, an improved conjugation method would be desirable.

SUMMARY

In a first aspect, a method of conjugating a substrate includesexchanging a counter ion associated with a biomolecule with a lipophiliccounter ion to form a biomolecule complex, dispersing the biomoleculecomplex in a nonaqueous solvent, and coupling the biomolecule complex toa substrate in the presence of the nonaqueous solvent.

In a second aspect, a method of conjugating a polymeric particleincludes exchanging a cationic counter ion associated with apolynucleotide with a lipophilic cationic counter ion to form apolynucleotide complex, the polynucleotide including a nucleophilic oran electrophilic reactive group. The method further includes dispersingthe polynucleotide complex in a non-reactive, nonaqueous solvent andcoupling the polynucleotide to the polymeric particle by nucleophilic orelectrophilic substitution, the polymeric particle including anelectrophilic group or a nucleophilic group.

In a third aspect, a polymeric particle includes a polymer conjugated toa polynucleotide using the method of any one of above aspects orexamples.

In a fourth aspect, a method of sequencing includes amplifying a targetpolynucleotide in the presence of an oligonucleotide conjugatedpolymeric particle to form the polynucleotide conjugated polymericparticle. The oligonucleotide is at least partially complementary to thetarget polynucleotide. The oligonucleotide conjugated polymeric particleis formed by the method of any of the above aspects or examples. Themethod further includes applying the polynucleotide conjugated polymericparticle to a sequencing device, applying a primer to the polynucleotideconjugated polymeric particle, incorporating a nucleotide, and detectingthe incorporating.

In a fifth aspect, a method of isolating a target polynucleotideincludes contacting a first solution including the target polynucleotidewith a probe conjugated substrate. The probe of the probe conjugatedsubstrate is at least partially complementary to the targetpolynucleotide. The probe conjugated substrate is formed by the methodof any of the above aspects or examples. The method further includeswashing the probe conjugated substrate while the target polynucleotideis coupled to the probe and releasing the target polynucleotide in asecond solution.

In an additional aspect, a method of conjugating an oligonucleotide to apolymer includes treating a polymer comprising amine functionality witha bis-NHS ester or a disuccinimidyl carbonate to form a functionalizedpolymer and treating the functionalized polymer with an amine terminatedoligonucleotide to form a conjugated polymer including theoligonucleotide.

BRIEF DESCRIPTION OF THE DRAWINGS

The present disclosure may be better understood, and its numerousfeatures and advantages made apparent to those skilled in the art byreferencing the accompanying drawings.

FIG. 1 includes an illustration of an exemplary method for treatingpolynucleotides.

FIG. 2 includes an illustration of an exemplary conjugation method.

FIG. 3 includes an illustration of an exemplary sequencing method.

FIG. 4 includes an illustration of an exemplary direct conjugationmethod.

FIG. 5 includes an illustration of an exemplary indirect conjugationmethod.

FIG. 6 includes an illustration of an exemplary conjugation method.

The use of the same reference symbols in different drawings indicatessimilar or identical items.

DETAILED DESCRIPTION

In an embodiment, a method of conjugation includes exchanging counterions associated with a biomolecule with lipophilic counter ions to forma biomolecule complex. In an example the counter ions are cationic. Thebiomolecule complex, such as a polynucleotide complex, can be dispersedin a nonaqueous solvent and conjugated to a substrate in the presence ofthe nonaqueous solvent. An exemplary substrate includes a surface, suchas a ceramic, metallic, or polymeric surface. In another example, asubstrate includes a swellable polymeric particle. In particular, thebiomolecule can be conjugated to functional groups of the swellablepolymer throughout the polymeric particle.

In example, the conjugated substrate can be used to capture a targetpolynucleotide. In a particular example, the conjugated polynucleotidecan be extended based on the captured target polynucleotide. In the caseof a polymeric particle including multiple copies of the conjugatedpolynucleotide, copies of the conjugated polynucleotide can be extendedin accordance with a target polynucleotide to provide multiple copies ofa complement to the target polynucleotide. Such a particle can be usefulin sequencing techniques, such as ion or pH-based sequencing techniques.

In particular, the present method performs conjugation within anonaqueous solution. It has been discovered that water can compete withor interfere with various conjugation chemistries, reducing conjugationefficiency. For example, water can interfere with nucleophilic orelectrophilic substitution. Water can compete with the nucleophile forthe electrophile by hydrolyzing the electrophile to an inactive moietyfor conjugation. By conjugating in a nonaqueous solution or solvent,conjugation methods, such as nucleophilic or electrophilic substitution,become more efficient. In addition, new conjugation chemistries can beutilized in a nonaqueous environment.

A biomolecule can include nucleosides, nucleotides, nucleic acids(oligonucleotides and polynucleotides), polypeptides, saccharides,polysaccharides, lipids, and derivatives or analogs thereof. In aparticular example, the biomolecule is a polypeptide or a nucleic acid,such as a polynucleotide. For example, the biomolecule can be apolynucleotide or an analog thereof.

As illustrated in FIG. 1, counter ions of a biomolecule can be exchangedwith lipophilic counter ions to provide a biomolecule complex that ismore lipophilic. The illustrated biomolecule is a polynucleotide 102. Asillustrated, the polynucleotide 102 is formed of a plurality ofpolymerized nucleotides. The carbohydrate moiety (X) of a nucleotide isbound to a phosphate group of a neighboring nucleotide. Each phosphategroup is associated with a cationic counter ion (M). In an example, thecationic counter ion (M) can be a metal ion. In another example, thecationic counter ion (M) can be ammonium or proton. In addition, thepolynucleotide 102 can include a linker group (L), linking a reactivegroup (P) to the nucleotide chain. Alternatively, the biomolecule can bea polynucleotide analog having a similar linker/reactive groupstructure, and the polynucleotide can have the reactive group (P)extending off one or more of the bases in addition to or instead of offthe carbohydrate (X).

In an example, the linker group (L) includes a hydrocarbon, an ether orpolyether group, or a combination thereof. The reactive group (P) canfunction to react with functional groups formed on a substrate, such asa polymeric substrate. In a particular example, the reactive group (P)can be an amine, thiol, maleimide, acetylene, azide, or a combinationthereof. For example, the reactive group (P) can be an amine or a thiol.In particular, the reactive group (P) can be an amine. In an example,the reactive group (P) can be a maleimide. In a further example, thereactive group (P) can be acetylene. In a further example, the reactivegroup (P) can be an azide.

The polynucleotide 102 is exposed to a lipophilic counter ion 104. Thelipophilic counter ion 104 can include a positively charged member (Y)coupled to one or more hydrocarbon groups (R1, R2, R3, R4) andassociated with an opposing ion (Z). In an example, the positivelycharged member (Y) can be nitrogen, phosphorus, sulfur, arsenic, or anycombination thereof. In particular, the positively charged member (Y) isnitrogen, phosphorous, sulfur, or a combination thereof. For example,the positively charged member (Y) can be a nitrogen or phosphorous. Inparticular, the positively charged member (Y) is nitrogen, forming anamine with hydrocarbon groups (R1, R2, R3, or R4).

The positively charged member (Y) includes one or more hydrocarbongroups, such as at least two hydrocarbon groups, at least threehydrocarbon groups, or at least four hydrocarbon groups. As illustrated,the positively charged member (Y) includes four hydrocarbon groups (R1,R2, R3, or R4). The hydrocarbon groups (R1, R2, R3, or R4) independentlycan be an alkyl group, an aryl group, ether derivatives thereof, orcombinations thereof. In an example, an alkyl hydrocarbon group caninclude a methyl, ethyl, propyl, or butyl group, an ether derivativethereof, or a combination thereof. For example, the propyl can be ann-propyl, an iso-propyl, or a combination thereof. In an example, thebutyl group can be an n- butyl, isobutyl, sec-butyl, tert-butyl, or anycombination thereof. An exemplary aryl group can include a phenyl,tolyl, xylyl, or poly-aryl, such as naphthyl, ether derivatives thereof,or any combination thereof.

In particular, the lipophilic group [Y(R1)(R2)(R3)(R4)] can include alipophilic ammonium ion, a lipophilic phosphonium ion, a lipophilicarsonium ion, a lipophilic sulfonium ion, or a combination thereof. Anexemplary lipophilic ammonium ion includes a tetraalkylammonium, atetraarylammonium, mixed alkyl and aryl ammonium, or a combinationthereof. For example, an exemplary lipophilic ammonium ion is selectedfrom the group consisting of tetramethylamonium, tetraethylamonium,tetrapropylamonium, tetrabutylamonium, tetrapentylamonium,tetrahexylamonium, tetraheptylamonium, tetraoctylamonium, alkyl and arylmixtures thereof, and a combination thereof. An exemplary lipophilicphosphonium ion includes tetraphenylphosphonium. An exemplary lipophilicarsonium ion is a tetraalkylarsonium, a tetraarylarsonium, a mixed alkyland aryl arsonium ion, or a combination thereof. For example, thelipophilic arsonium ion is tetraphenylarsonium. An exemplary lipophilicsulfonium ion is a trialkylsulfonium ion. The ion (Z) can be an ion ofopposite charge to the lipophilic group [Y(R1)(R2)(R3)(R4)], such as ahydroxide, a halogen, a nitrate, a carbonate, a sulfate, a perchlorate,a phenolate, a tetraalkylborate, a tetraarylborate, a phosphate ion, orany combination thereof.

As a result of the exchange, the polynucleotide complex 106 exhibitslipophilic behavior and can be dispersed in a nonaqueous solvent. In anexample, the nonaqueous solvent is polar. In a further example, thenonaqueous solvent is not reactive with coupling groups on the substrateor functional groups of the polymer, such as the reactive group (P) ofthe polynucleotide complex 106. In an example, the solvent includes anamide, a urea, a carbonate, an ether, a sulfoxide, a sulfone, a hinderedalcohol, or a combination thereof. An exemplary amide or urea includesformamide, N,N-dimethylformamide, acetamide, N,N-dimethylacetamide,hexamethylphosphoramide, pyrrolidone, N-methylpyrrolidone,N,N,N′,N′-tetramethylurea, N,N′-dimethyl-N,N′-trimethyleneurea, or acombination thereof. An exemplary carbonate includes dimethyl carbonate,propylene carbonate, or a combination thereof. An exemplary etherincludes tetrahydrofuran. An exemplary sulfoxide or sulfone includesdimethylsulfoxide, dimethylsulfone, or a combination thereof. Anexemplary hindered alcohol includes tert-butyl alcohol.

Following the exchange or as part of the exchange, the polynucleotidecomplex 106 can be dispersed in the nonaqueous solvent. The dispersedpolynucleotide complex 106 can be used in conjugation of a substrate.

The substrate can be a solid surface, a particle, or a combinationthereof. In an example, the substrate can be a solid surface. Thesubstrate can include planar, concave, or convex surfaces, or anycombination thereof. A substrate can comprise texture or features,including etching, cavitation or bumps. Alternatively, the substrate canlack any texture or features. The substrate can include capillarystructures, channels, grooves, wells or reservoirs. In an example, thesubstrate can be a mesh. The substrate can be porous, semi-porous ornon-porous. In a further example, the substrate can be a filter or gel.The substrate can include the top of a pin (e.g., pin arrays). Thesubstrate may be made from materials such as glass, borosilicate glass,silica, quartz, fused quartz, mica, polyacrylamide and N-substitutedpolyacrylamides, plastic polystyrene, polycarbonate, polymethacrylate(PMA), polymethyl methacrylate (PMMA), polydimethylsiloxane (PDMS),silicon, germanium, graphite, ceramics, silicon, semiconductor, highrefractive index dielectrics, crystals, gels, polymers, or films (e.g.,films of gold, silver, aluminum, or diamond). In particular, thesubstrate can include a solid substrate having a metal film or metalcoating.

In a particular example, a substrate includes polymeric particles havingporosity that permits the diffusion of the polynucleotide complex 106within the polymeric particle. In particular, the polymeric particle caninclude reactive functional groups that are reactive to the reactivegroup (P) of the polynucleotide complex 106. In an example, thepolymeric particles are hydrophilic. The polymeric particle can beswellable. For example, the polymeric particle can be a hydrogel. Thehydrophilic polymeric particle can expose functional groups, such ashydroxyl groups or amine groups. Such groups can be replaced with otherfunctional groups to facilitate conjugation with the polynucleotidecomplex 106.

In an example, the polymeric particle can be formed of styrenicpolymers, acrylate polymers, acrylamide polymers, or a combinationthereof. For example, the polymeric particle can be formed frompolymerized monomers. The monomer can be a radically polymerizablemonomer, such as a vinyl-based monomer. In particular, the monomer caninclude a hydrophilic monomer. In an example, the hydrophilic monomercan include acrylamide, vinyl acetate, hydroxyalkylmethacrylate, or anycombination thereof. In a particular example, the hydrophilic monomer isan acrylamide, such as an acrylamide including hydroxyl groups, aminogroups, or a combination thereof. In an example, the hydrophilic monomeris an aminoalkyl acrylamide. In another example, the acrylamide can be ahydroxyalkyl acrylamide, such as hydroxyethyl acrylamide. In particular,the hydroxyalkyl acrylamide can includeN-[tris(hydroxymethyl)methyl)acrylamide, N-(hydroxymethyl)acrylamide, ora combination thereof. In a particular example, the monomer includeshydroxyl groups. In a further example, a comonomer can be included, suchas an aminoacrylamide, for example, an acrylamide functionalized with anamine terminated polyethylene glycol or acrylopiperazine.

In an example, the polymer of the polymeric particle can be formed frompolymerizing a monomer and a crosslinker. In an example, the crosslinkeris included in a mass ratio of monomer to crosslinker in a range of 15:1to 1:2, such as a range of 10:1 to 1:1, a range of 6:1 to 1:1, or even arange of 4:1 to 1:1. In particular, the crosslinker can be a divinylcrosslinker. For example, a divinyl crosslinker can include adiacrylamide, such as N,N′-(ethane-1,2-diyl)bis(2-hydroxylethyl)acrylamide, N,N′-(2-hydroxypropane-1,3-diyl)diacrylamide, or acombination thereof. In another example, a divinyl crosslinker includesethyleneglycol dimethacrylate, divinylbenzene, hexamethylenebisacrylamide, trimethylolpropane trimethacrylate, ethylenedimethacrylate, or a combination thereof.

In an example, the polymeric particle can be a hydrophilic particle,such as a hydrogel particle. A hydrogel is a polymer that can absorb atleast 20% of its weight in water, such as at least 45%, at least 65%, atleast 85%, at least 100%, at least 300%, at least 1000%, at least 1500%or even at least 2000% of its weight in water.

The polymeric particles can have a desirable particle size, such as aparticle size not greater than 100 μm, not greater than 30 μm, or notgreater than 3 μm. The average particle size is the mean particlediameter. For example, the average particle size may be not greater than2 μm, such as not greater than 1.5 μm, not greater than 1.1 μm, notgreater than 0.8 μm, not greater than 0.6 μm, not greater than 0.5 μm,or even not greater than 0.3 μm. In a particular example, the averageparticle size can be in a range of 0.1 μm to 100 μm, such as a range of0.1 μm to 50 μm or a range of 0.1 μm to 1.1 μm.

In a further example, the plurality of particles is monodisperse and mayhave a desirably low coefficient of variance, such as a coefficient ofvariance of not greater than 20%. The coefficient of variance (CV) isdefined as 100 times the standard deviation divided by average, where“average” is the mean particle diameter and standard deviation is thestandard deviation in particle size. The “average” alternatively can beeither the z-average or mode particle diameter. In accordance with usualpractice, CV is calculated on the main mode, i.e., the main peak,thereby excluding minor peaks relating to aggregates. Thus, someparticles below or above mode size may be discounted in the calculationwhich may, for example, be based on about 90% of total particle numberof detectable particles. Such a determination of CV is performable on aCPS disc centrifuge or a coulter counter. For example, the coefficientof variance (CV) of the polymeric particles may be not greater than 15%,such as not greater than 10%, not greater than 5%, not greater than4.5%, not greater than 4.0%, not greater than 3.5%, or even not greaterthan 3.0%.

In a further example, a hydrophilic polymeric particle in water can benot greater than 50 wt % polymer, such as not greater than 30 wt %polymer, not greater than 20 wt % polymer, not greater than 10 wt %polymer, not greater than 5 wt % polymer, or even not greater than 2 wt% polymer.

In an additional example, the polymeric particle can have a porositypermitting diffusion of polynucleotides, proteins or enzymes. In anexample, the polymeric particles can have a porosity to permit diffusionof proteins having a size of at least 50 kilodaltons, such as at least100 kilodaltons, at least 200 kilodaltons, at least 250 kilodaltons, oreven at least 350 kilodaltons.

Whether a solid surface or a polymeric particle, such as the polymericparticles described above, the polynucleotide complex 106 illustrated inFIG. 1 can be conjugated to a substrate in the presence of a nonaqueoussolvent. For example, a polymer of a substrate can include hydroxylgroups. A portion of the hydroxyl groups can be replaced withalternative functional groups to facilitate conjugation with apolynucleotide complex, such as the polynucleotide complex 106illustrated in FIG. 1.

A substrate can undergo direct conjugation or indirect conjugation. In aparticular example, the functional groups of the polymer, such as ahydroxyl group, can undergo nucleophilic or electrophilic substitutionto conjugate the polymer to a polynucleotide. For example, the couplinggroup of a substrate can include one of a nucleophile or an electrophileand the terminal reactive group of the polynucleotide can include theother of the nucleophile or the electrophile. The polynucleotide can becoupled to the polymeric particle by nucleophilic or electrophilicsubstitution. In an example, direct conjugation of a polymer including ahydroxyl group can include replacing the hydroxyl group with a sulfonateester that is subsequently conjugated to a oligonucleotide orpolynucleotide including an amine or a thiol reactive group. Forexample, hydroxyl groups on a polymeric particle can be activated byreplacing at least a portion of the hydroxyl groups with a sulfonateester group. Exemplary sulfonate ester groups can be derived fromtresyl, mesyl, tosyl, or fosyl chloride, or any combination thereof, toprovide the sulfonate ester functional group in place of at least aportion of the hydroxyl groups. Sulfonate esters can act to permitnucleophiles to replace the sulfonate ester. The sulfonate ester mayfurther react with liberated chlorine to provide chlorinated groups thatcan be used in a process to conjugate the particles. Alternatively,hydroxyl groups can be replaced with halogen groups which are reactivewith the reactive groups of a polynucleotide complex. In a furtherexample, amine groups of comonomers of the polymeric polymer can bereactive with reactive groups of the polynucleotide complex.

Alternatively, the conjugation may be indirect in which polymerfunctional groups, such as hydroxyl groups, are replaced through aseries of substitutions, resulting in a functional group (couplinggroup) that can be reacted with a reactive group of a polynucleotidecomplex. For example, FIG. 2 illustrates a polymer 202 that includeshydroxyl groups, which can be substituted with functional groups througha series of one or more substitutions.

For example, at 204, at least a portion of the hydroxyl groups can bereplaced with a functional group (A), such as a sulfonate ester, ahalogen other than fluoride, or a combination thereof. Such functionalgroups (A) can further be replaced, as illustrated at 206, with afunctional group (B), such as an azide, phthalimide, thioester,N-protected diamine, N-protected aminothioether, amino(oligonucleotide),or a combination thereof. Optionally, such functional groups (B) can befurther activated, as illustrated at 208, with a functional group (C),such as an amine, thiol, or combination thereof. Moreover, suchfunctional groups (C), illustrated at 208, can be replaced withfunctional groups (D), as illustrated at 210, such as monoamide-mono(NHSester) dicarboxylic acid, succinimide thioether(oligonucleotide), or acombination thereof. Even further, the functional group (D) illustratedat 210 can be replaced, as illustrated at 212, with a functional group(E), such as a monoamide-(amino-oligonucleotide)-monoamide dicarboxylicacid. As such, the functional groups of the original polymer can undergoone or more of a series of substitutions providing reaction sitessuitable for reacting with a polynucleotide complex.

Alternatively, an acrylamide polymer having amine functionality can beused in place of the polymer including hydroxyl groups. For example, theillustrated hydroxyl groups can be replaced with an amine terminatedpolyether (e.g., amine terminated PEG) group. With amine functionality,the illustrated method can begin, for example, at (C).

In a particular example, the functional group (A) at 204 can be asulfonate ester, a halogen, or a combination thereof. In particular, thehalogen is a halogen other than fluorine, such as chlorine. Thefunctional group (A) can react with the polynucleotide complex 106 wherethe linker group (L) is a hydrocarbon or polyether and the reactivegroup (P) is an amine in a non-aqueous solvent to yield functional group(B) at 206, such as an amino(oligonucleotide). In another example inwhich the functional group (A) at 204 is replaced by functional group(B) at 206, the functional group (A) can be a sulfonate ester, halogen,or a combination thereof, and the functional group (B) can be in anazide, phthalimide, mono(N-protected)-diamine, or N-protectedaminothioether. The functional group (B) can be further activated to afunctional group (C), such as an amine. The amine functional group (C)can be further activated with a functional group (D), such as a(mono-amide)-(mono-NHS ester) dicarboxylic acid. The functional group(D) can be further substituted with functional group (E), such as an(amino(oligonucleotide))-dicarboxylic acid by reacting with thepolynucleotide complex 106 that includes a linking group (L) ofhydrocarbon or polyether and a reactive functional group (P) of anamine, in a non-aqueous solvent. Alternatively, the polymer particle canbe a copolymer including a comonomer that includes amine functionalgroups. Such amine functional groups can be reactive with

In a further example, the functional group (A) is a sulfonate ester, ahalogen, or combination thereof. The functional group (B) is an azide.In such a case, the functional group (B) can be substituted with afunctional group (C), such as an (oligonucleotide)triazole. Thepolynucleotide complex includes a linker group (L) of a hydrocarbon orpolyether and a reactive functional group (P) of acetylene.

In an additional example, the functional group (A) can be a sulfonateester, halogen, or a combination thereof. The functional group (A) issubstituted with a functional group (B), such as a thioester. Thethioester functional group (B) can be substituted with a functionalgroup (C), such as a thiol, which is further substituted by thefunctional group (D), such as a succinimide thioether(oligonucleotide).In such an example, the polynucleotide complex can include a linker (L),such as a hydrocarbon or polyether, and a reactive group (P), such as amaleimide.

Other activation chemistries include incorporating multiple steps toconvert a specified functional group to accommodate specific desiredlinkages. For example, the sulfonate modified hydroxyl group can beconverted into a nucleophilic group through several methods. In anexample, reaction of the sulfonate ester with azide anion yields anazide substituted hydrophilic polymer. The azide can be used directly toconjugate to an acetylene substituted biomolecule via “CLICK” chemistrythat can be performed with or without copper catalysis. Optionally, theazide can be converted to amine by, for example, catalytic reductionwith hydrogen or reduction with an organic phosphine. The resultingamine can then be converted to an electrophilic group with a variety ofreagents, such as di-isocyanates, bis-NHS esters, cyanuric chloride, ora combination thereof. In an example, using di-isocyanates yields a urealinkage between the polymer and a linker that results in a residualisocyanate group that is capable of reacting with an amino substitutedbiomolecule to yield a urea linkage between the linker and thebiomolecule. In another example, using bis-NHS esters yields an amidelinkage between the polymer and the linker and a residual NHS estergroup that is capable of reacting with an amino substituted biomoleculeto yield an amide linkage between the linker and the biomolecule. Anexemplary bis-NHS ester includes bis-succinimidyl C2-C12 alkyl esters,such as bis-succinimidyl suberate or bis-succinimidyl glutarate. In afurther example, using cyanuric chloride yields an amino-triazinelinkage between the polymer and the linker and two residualchloro-triazine groups one of which is capable of reacting with an aminosubstituted biomolecule to yield an amino-triazine linkage between thelinker and the biomolecule. Other nucleophilic groups can beincorporated into the particle via sulfonate activation. For example,reaction of sulfonated particles with thiobenzoic acid anion andhydrolysis of the consequent thiobenzoate incorporates a thiol into theparticle which can be subsequently reacted with a maleimide substitutedbiomolecule to yield a thio-succinimide linkage to the biomolecule.Thiol can also be reacted with a bromo-acetyl group.

In a further example, a polymer particle can be treated to form aminefunctionality that can be conjugated through the use a bis-NHS ester ora disuccinimidyl carbonate. An exemplary bis-NHS ester includesbis-succinimidyl C2-C12 alkyl esters, such as bis-succinimidyl suberateor bis-succinimidyl glutarate. For example, a polymeric particle, suchas a particle including exposed hydroxyl groups, can be treated toreplace the hydroxyl groups with a halogent, such as chlorine. Theparticle can be further treated under acid conditions with a tBOC(N-tert-butoxycarbonyl) protected amine terminated polyether, such aspolyethylene glycol (PEG), providing amine functionality that can befurther used to conjugate the polymer particle. Alternatively, thepolymer particle can be polymerized using monomers that provide theamine functionality. The polymer particle including the amine group canbe further treated using a bis-NHS ester or a disuccinimidyl carbonate,which is further exposed to an amine terminated oligonucleotide,resulting a polymer particle conjugated to the oligonucleotide.

Following conjugation, the polymeric particle can include a density ofpolynucleotides, termed nucleotide density, of at least 7×10⁴ per μm³.For example, the nucleotide density can be at least 10⁵ per μm³, such asat least 10⁶ per μm³, at least 2×10⁶ per μm³, at least 5×10⁶ per μm³, atleast 8×10⁶ per μm³, at least 1×10⁷ per μm³, or even at least 3×10⁷ perμm³.

In a particular example, the conjugated particles can be used inseparation techniques or sequencing techniques. For example, theconjugated particles can be used to capture target polynucleotides. Inan example, the polynucleotides conjugated to the polymer can beextended based on captured target polynucleotides. Such conjugatedparticles can be use in sequencing techniques, such as a ion-based orpH-based sequencing techniques. As illustrated in FIG. 3, a plurality ofconjugated polymeric particles 304 can be placed in a solution alongwith a plurality of target polynucleotides 302. The plurality ofparticles 304 can be conjugated with probe polynucleotides to bind withtarget polynucleotides 302. For example, the conjugated particles 304can include an oligonucleotide complementary to a portion of the targetpolynucleotides 302.

In a particular embodiment, the particles 304 and polynucleotides 302are subjected to polymerase chain reaction (PCR) amplification. Forexample, dispersed phase droplets 306 or 308 are formed as part of anemulsion and can include a hydrophilic particle or a polynucleotide. Inan example, the target polynucleotides 302 and the hydrophilic particles304 are provided in low concentrations and ratios relative to each othersuch that a single polynucleotide 302 is likely to reside within thesame dispersed phase droplets as a single hydrophilic particle 304.Other droplets, such as a droplet 308, can include a single hydrophilicparticle and no polynucleotide. Each droplet 306 or 308 can includeenzymes, nucleotides, salts or other components sufficient to facilitateduplication of the polynucleotide.

Duplication of the target polynucleotide can include modulating theduplication conditions. Modulating can optionally include: increasing ordecreasing the polymerase concentration; increasing or decreasing thenucleotide concentration; increasing or decreasing a cationconcentration; increasing or decreasing a reaction temperature, time orpH, and the like. Modulating can include increasing or decreasing therate of the reaction, increasing or decreasing the yield of product ofthe reaction, and the like. Duplication can be performed in the presenceof appropriate buffers or nucleotides (including nucleotide analogs orbiotinylated nucleotides).

In particular, the polynucleotide to be amplified can be captured by thepolymeric particle. Exemplary methods for capturing nucleic acid caninclude: hybridizing a polynucleotide to an oligonucleotide that isattached to a polymeric particle. Methods for capturing nucleic acidscan include: (a) providing a polymeric particle attached to asingle-stranded oligonucleotide (e.g., a capture oligonucleotide); (b)providing a single-stranded polynucleotide; and (c) hybridizing thesingle-stranded oligonucleotide to the single-stranded polynucleotides,thereby capturing the single-stranded polynucleotide to the polymericparticle. Each of the polymeric particles can be attached with aplurality of single-stranded oligonucleotides (e.g., captureoligonucleotides). In some embodiments, step (c) can be conducted with aplurality of single-stranded polynucleotides. In some embodiments, atleast a portion of the single-stranded oligonucleotide includes anucleotide sequence that is complementary (or partially complementary)to at least a portion of the single-stranded polynucleotide.

In an example, the method can further include amplifying thepolynucleotide into a plurality of polynucleotides and attaching atleast a portion of the plurality of polynucleotides to the hydrophilicparticle, thereby generating a hydrophilic particle including aplurality of attached polynucleotides. Alternatively, the method caninclude amplifying the polynucleotide into a plurality of complementarypolynucleotides by extending the conjugated oligonucleotide, therebygenerating a hydrogel particle including a plurality of attachedpolynucleotides.

In a further example, methods for nucleotide incorporation can include:conducting a nucleotide polymerization reaction on a polynucleotide thatis hybridized to an oligonucleotide that is attached to a polymericparticle. In some embodiments, methods for nucleotide incorporationcomprise: (a) providing a polymeric particle attached to asingle-stranded oligonucleotide (e.g., a primer oligonucleotide); (b)providing a single-stranded template polynucleotide; (c) hybridizing thesingle-stranded oligonucleotide to the single-stranded templatepolynucleotide; and (d) contacting the single-stranded templatepolynucleotide with a polymerase and at least one nucleotide underconditions suitable for the polymerase to catalyze polymerization of atleast one nucleotide onto the single-stranded oligonucleotide, therebyconducting nucleotide incorporation. In some embodiments, each of thepolymeric particles can be attached with a plurality of single-strandedoligonucleotides (e.g., capture oligonucleotides). In some embodiments,steps (b), (c) or (d) can be conducted with a plurality ofsingle-stranded polynucleotides. In some embodiments, at least a portionof the single-stranded oligonucleotide comprises a nucleotide sequencethat is complementary (or partially complementary) to at least a portionof the single-stranded polynucleotide. In some embodiments, a systemcomprises a single-stranded polynucleotide hybridized to asingle-stranded oligonucleotide which is attached to a polymericparticle, wherein at least one nucleotide is polymerized onto the end ofthe single-stranded oligonucleotide.

In another example, methods for primer extension can include: conductinga primer extension reaction on a polynucleotide that is hybridized to anoligonucleotide that is attached to a polymeric particle. In someembodiments, methods for nucleic acid primer extension comprise: (a)providing a polymeric particle attached to a single-strandedoligonucleotide (e.g., a primer oligonucleotide); (b) providing asingle-stranded template polynucleotide; (c) hybridizing thesingle-stranded oligonucleotide to the single-stranded templatepolynucleotide; and (d) contacting the single-stranded templatepolynucleotide with a polymerase and at least one nucleotide underconditions suitable for the polymerase to catalyze polymerization of atleast one nucleotide onto the single-stranded oligonucleotide, therebyextending the primer. In some embodiments, each of the polymericparticles can be attached with a plurality of single-strandedoligonucleotides (e.g., capture oligonucleotides). In some embodiments,step (b), (c) or (d) can be conducted with a plurality ofsingle-stranded polynucleotides. In some embodiments, at least a portionof the single-stranded oligonucleotide comprises a nucleotide sequencethat is complementary (or partially complementary) to at least a portionof the single-stranded polynucleotide. In some embodiments, a systemcomprises a single-stranded polynucleotide hybridized to asingle-stranded oligonucleotide which is attached to a polymericparticle, wherein the single-stranded oligonucleotide is extended withone or more nucleotides.

In additional examples, methods for nucleic acid amplification comprise:conducting a primer extension reaction on a polynucleotide that ishybridized to an oligonucleotide which is attached to a polymericparticle. In some embodiments, methods for nucleic acid amplificationcomprise: (a) providing a polymeric particle attached to asingle-stranded oligonucleotide (e.g., a primer oligonucleotide); (b)providing a single-stranded template polynucleotide; (c) hybridizing thesingle-stranded oligonucleotide to the single-stranded templatepolynucleotide; (d) contacting the single-stranded templatepolynucleotide with a polymerase and at least one nucleotide underconditions suitable for the polymerase to catalyze polymerization of atleast one nucleotide onto the single-stranded oligonucleotide so as togenerate an extended single-stranded oligonucleotide. In someembodiments, the method further comprises: (e) removing (e.g.,denaturing) the single-stranded template polynucleotide from theextended single-stranded oligonucleotide so that the single-strandedoligonucleotide remains attached to the polymeric particle; (f)hybridizing the remaining single-stranded oligonucleotide to a secondsingle-stranded template polynucleotide; and (g) contacting the secondsingle-stranded template polynucleotide with a second polymerase and asecond at least one nucleotide, under conditions suitable for the secondpolymerase to catalyze polymerization of the second at least onenucleotide onto the single-stranded oligonucleotide so as to generate asubsequent extended single-stranded oligonucleotide. In someembodiments, steps (e), (f) and (g) can be repeated at least once. Insome embodiments, the polymerase and the second polymerase comprise athermostable polymerase. In some embodiments, the conditions suitablefor nucleotide polymerization include conducting the nucleotidepolymerization steps (e.g., steps (d) or (g)) at an elevatedtemperature. In some embodiments, the conditions suitable for nucleotidepolymerization include conducting the nucleotide polymerization step(e.g., steps (d) or (g)) at alternating temperatures (e.g., an elevatedtemperature and a relatively lower temperature). In some embodiments,the alternating temperature ranges from 60-95° C. In some embodiments,the temperature cycles can be about 10 seconds to about 5 minutes, orabout 10 minutes, or about 15 minutes, or longer. In some embodiments,methods for nucleic acid amplification can generate one or morepolymeric particles each attached to a plurality of templatepolynucleotides comprising sequences that are complementary to thesingle-stranded template polynucleotide or to the second single-strandedtemplate polynucleotide. In some embodiments, each of the polymericparticles can be attached with a plurality of single-strandedoligonucleotides (e.g., capture oligonucleotides). In some embodiments,step (b), (c), (d), (e), (f) or (g) can be conducted with a plurality ofsingle-stranded polynucleotides. In some embodiments, at least a portionof the single-stranded oligonucleotide comprises a nucleotide sequencethat is complementary (or partially complementary) to at least a portionof the single-stranded polynucleotide. In some embodiments, methods fornucleic acid amplification (as described above) can be conducted in anaqueous phase solution in an oil phase (e.g., dispersed phase droplet).

Following PCR, particles are formed, such as particle 310, which caninclude the hydrophilic particle 312 and a plurality of copies 314 ofthe target polynucleotide or complements thereof. While thepolynucleotides 314 are illustrated as being on a surface of theparticle 310, the polynucleotides 314 can extend within the particle310. Hydrogel and hydrophilic particles having a low concentration ofpolymer relative to water can include polynucleotide segments on theinterior of and throughout the particle 310 or polynucleotides canreside in pores and other openings. In particular, the particle 310 canpermit diffusion of enzymes, nucleotides, primers and reaction productsused to monitor the reaction. A high number of polynucleotides perparticle produces a better signal in particular sequencing techniques.

In an exemplary embodiment, the particle 310 can be utilized in asequencing device. For example, a sequencing device 316 can include anarray of wells 318. A particle 310 can be placed within a well 318.

In an example, a primer can be added to the wells 318 or the particle310 can be pre-exposed to the primer prior to placement in the well 318.The primer and polynucleotide form a nucleic acid duplex including thepolynucleotide (e.g., a template nucleic acid) hybridized to the primer.The nucleic acid duplex is an at least partially double-strandedpolynucleotide. Enzymes and nucleotides can be provided to the well 318to facilitate detectible reactions, such as nucleotide incorporation.

Sequencing can be performed by detecting nucleotide addition. Nucleotideaddition can be detected using methods such as fluorescent emissionmethods or ion detection methods. For example, a set of fluorescentlylabeled nucleotides can be provided to the system 316 and can migrate tothe well 318. Excitation energy can be also provided to the well 318.When a nucleotide is captured by a polymerase and added to the end of anextending primer, a label of the nucleotide can fluoresce, indicatingwhich type of nucleotide is added.

In an alternative example, solutions including a single type ofnucleotide can be fed sequentially. In response to nucleotide addition,the pH within the local environment of the well 318 can change. Such achange in pH can be detected by ion sensitive field effect transistors(ISFET). As such, a change in pH can be used to generate a signalindicating the order of nucleotides complementary to the polynucleotide314 of the particle 310.

In particular, a sequencing system can include a well, or a plurality ofwells, disposed over a sensor pad of an ionic sensor, such as a fieldeffect transistor (FET). In some embodiments, a system includes one ormore polymeric particles loaded into a well which is disposed over asensor pad of an ionic sensor (e.g., FET), or one or more polymericparticles loaded into a plurality of wells which are disposed oversensor pads of ionic sensors (e.g., FET). In some embodiments, an FETcan be a chemFET or an ISFET. A “chemFET” or chemical field-effecttransistor, includes a type of field effect transistor that acts as achemical sensor. It is the structural analog of a MOSFET transistor,where the charge on the gate electrode is applied by a chemical process.An “ISFET” or ion-sensitive field-effect transistor can be used formeasuring ion concentrations in solution; when the ion concentration(such as H+) changes, the current through the transistor changesaccordingly.

The FET may be a FET array. As used herein, an “array” is a planararrangement of elements such as sensors or wells. The array may be oneor two dimensional. A one dimensional array can be an array having onecolumn (or row) of elements in the first dimension and a plurality ofrows (or columns) in the second dimension. The number of columns (orrows) in the first and second dimensions may or may not be the same.

One or more microfluidic structures can be fabricated above the FETsensor array to provide for containment or confinement of a biologicalor chemical reaction. For example, in one implementation, themicrofluidic structure(s) can be configured as one or more wells (ormicrowells, or reaction chambers, or reaction wells, as the terms areused interchangeably herein) disposed above one or more sensors of thearray, such that the one or more sensors over which a given well isdisposed detect and measure analyte presence, level, or concentration inthe given well. In some embodiments, there can be a 1:1 correspondenceof FET sensors and reaction wells.

Returning to FIG. 3, in another example, a well 318 of the array ofwells can be operatively connected to measuring devices. For example,for fluorescent emission methods, a well 318 can be operatively coupledto a light detection device. In the case of ionic detection, the lowersurface of the well 318 may be disposed over a sensor pad of an ionicsensor, such as a field effect transistor.

One exemplary system involving sequencing via detection of ionicbyproducts of nucleotide incorporation is the Ion Torrent PGM™ sequencer(Life Technologies), which is an ion-based sequencing system thatsequences nucleic acid templates by detecting hydrogen ions produced asa byproduct of nucleotide incorporation. Typically, hydrogen ions arereleased as byproducts of nucleotide incorporations occurring duringtemplate-dependent nucleic acid synthesis by a polymerase. The IonTorrent PGM™ sequencer detects the nucleotide incorporations bydetecting the hydrogen ion byproducts of the nucleotide incorporations.The Ion Torrent PGM™ sequencer can include a plurality of templatepolynucleotides to be sequenced, each template disposed within arespective sequencing reaction well in an array. The wells of the arraycan each be coupled to at least one ion sensor that can detect therelease of H+ ions or changes in solution pH produced as a byproduct ofnucleotide incorporation. The ion sensor comprises a field effecttransistor (FET) coupled to an ion-sensitive detection layer that cansense the presence of H+ ions or changes in solution pH. The ion sensorcan provide output signals indicative of nucleotide incorporation whichcan be represented as voltage changes whose magnitude correlates withthe H+ ion concentration in a respective well or reaction chamberDifferent nucleotide types can be flowed serially into the reactionchamber, and can be incorporated by the polymerase into an extendingprimer (or polymerization site) in an order determined by the sequenceof the template. Each nucleotide incorporation can be accompanied by therelease of H+ ions in the reaction well, along with a concomitant changein the localized pH. The release of H+ ions can be registered by the FETof the sensor, which produces signals indicating the occurrence of thenucleotide incorporation. Nucleotides that are not incorporated during aparticular nucleotide flow may not produce signals. The amplitude of thesignals from the FET can also be correlated with the number ofnucleotides of a particular type incorporated into the extending nucleicacid molecule thereby permitting homopolymer regions to be resolved.Thus, during a run of the sequencer multiple nucleotide flows into thereaction chamber along with incorporation monitoring across amultiplicity of wells or reaction chambers can permit the instrument toresolve the sequence of many nucleic acid templates simultaneously.Further details regarding the compositions, design and operation of theIon Torrent PGM™ sequencer can be found, for example, in U.S. patentapplication Ser. No. 12/002,781, now published as U.S. PatentPublication No. 2009/0026082; U.S. patent application Ser. No.12/474,897, now published as U.S. Patent Publication No. 2010/0137143;and U.S. patent application Ser. No. 12/492,844, now published as U.S.Patent Publication No. 2010/0282617, all of which applications areincorporated by reference herein in their entireties.

EXAMPLE Example 1

An oligonucleotide is directly conjugated to a mesyl activated Dynalparticle, as illustrated in FIG. 4. While functional groups areillustrated as being on the surface of the particle, functional groupscan exist throughout the particle. Mesyl chloride activated microgelsare prepared via the Ugelstad process, a seeded emulsion polymerization.Thus formed particles are washed in N-methyl-2-pyrrolidone (NMP) inpreparation for conjugation with ion exchanged single stranded DNA.

The sodium salt of 5′-NH2-C6-30-mer oligonucleotide was dissolved in 0.1M tetrabutylammonium acetate, and injected onto a reverse phase HPLCcolumn. Elution was performed with 0.1 M tetrabutylammonium acetatemobile phase. The fraction containing nucleic acid was collected,lyophilized to a dry powder, and re-suspended in dryN-methyl-2-pyrrolidone (NMP).

Five million particle (5.0×10⁹) are dispersed in 350 uL of anhydrous NMPand vortex mixed to disperse. 124 uL of Bu4NAc-DNA (5′-NH2-C6-30-meroligonucleotide) in NMP (4.10 mM) is directly added to the particlemixture. 19.5 uL of tetraethylammonium borate (26.14 mM) is then addedto the reaction mixture for a final volume of ˜500 uL.

The reaction mixture is quickly vortex mixed and is gently mixed at 70°C. for 16 hours. The mixture is centrifuged, the supernatant isdecanted, and the particles are re-suspended in 1 mL of NMP. Aftervortex mixing, the re-suspended microgel particles are pelleted with twocycles of precipitation/dispersion in NMP. After the second NMP wash,the pellets are brought up in 1 mL of 2×SSPE/0.1% sodium dodecyl sulfate(SDS), mixed and centrifuged to pellets. Finally, the particles arebrought up in 1 mL of 1×PBS/0.1% Triton X-100, mixed and centrifuged toa firm pellet, repeating this process three times. After the finalcycle, the conjugated microgels are re-suspended in 500 uL 1×PB S/0.1%Triton X-100.

The conjugated materials are then counted after labeling with SYBR-goldstain with a flow cytometer. The materials are ready for PCRamplification of DNA template in preparation for sequencing.

Example 2

A particle is conjugated through a series of substitutions asillustrated in FIG. 5. While functional groups are illustrated as beingon the surface of the particle, functional groups can exist throughoutthe particle.

To a solution of mesyl chloride activated hydrogels in NMP (2 billion, 1ml) in 2 ml centrifuge tube, a solution of saturated tetrabutylammoniumazide in NMP (800 uL) is added, and the reaction mixture is stirredovernight at 60° C. The reaction mixture is centrifuged at 21300 rcf for10 min and supernatant is removed. A resulting pellet is re-suspended inNMP and centrifuged to remove supernatant. The process is repeated twotimes.

A resulting pellet is re-suspended in de-ionized (DI) water (1 ml). Thereaction mixture is centrifuged at 21300 rcf for 10 min and supernatantis removed. The process is repeated 2 more times.

To the pellet, 1 ml of TCEP solution (DI water, 1M) is added and thereaction mixture is stirred at room temperature overnight. The reactionmixture is centrifuged to remove supernatant at 21300 rcf for 10 min.The resulting pellet is re-suspended in DI water and is centrifuged at21300 rcf for 10 min to remove supernatant two more times. The reactionmixture is re-suspended in anhydrous NMP (1 ml) and is centrifuged at21300 rcf for 10 min. The supernatant is removed, and the process isrepeated 3 more times.

The resulting pellet is re-suspended in anhydrous NMP (500 ul) and asolution of Bis-NHS suberate in anhydrous NMP (200 uL, 20 mg) is addedto the reaction mixture. The reaction mixture is stirred at 70° C.overnight. The reaction mixture is centrifuged to remove supernatant at21300 rcf for 10 min. The reaction mixture is re-suspended in anhydrousNMP (1 ml) and is centrifuged to remove supernatant at 21300 rcf for 10min. The process is repeated five more times, and a resulting pellet isre-suspended in anhydrous NMP (200 uL).

To the reaction mixture, a solution of oligonucleotide (Bu4NAc-DNA(5′-NH2-C6-30-mer oligonucleotide) in NMP) (0.6 micromols, 200 uL) and asolution of tetrabutylammonium borate in anhydrous NMP (60 uL, 1 mg in200 uL) is added, and the reaction mixture is stirred at 70° C.overnight.

The reaction mixture is centrifuged at 21300 rcf for 10 min to removesupernatant and a resulting pellet is re-suspended in NMP (1 ml). Thereaction mixture is centrifuged at 21300 rcf for 10 min and theresulting pellet is re-suspended in NMP. The process is repeated threemore times, and the pellet is re-suspended in 2×SSPE+0.2% SDS solution(1 ml) and is centrifuged at 21300 rcf for 10 min. The resulting pelletis re-suspended in 1×TE buffer with 0.1% Triton X100 (1 ml). Thereaction mixture is centrifuged at 21300 rcf for 10 min. The pellet isre-suspended in 1×TE buffer and stirred at 80° C. for 1 h. The mixtureis centrifuged at 21300 rcf for 10 min to remove the supernatant. Thepellet is re-suspended in 1×TE buffer with 0.1% Triton X100 (1 ml) andis centrifuge at 21300 rcf for 10 min to remove the supernatant. Theprocess is repeated once. The pellet is re-suspended in 30% ammoniasolution (1 ml) and is stirred at room temperature for 15 min. Thereaction mixture is centrifuged to remove the supernatant at 21300 rcffor 10 min, and the pellet is re-suspended in DI water (1 ml). Thereaction mixture is centrifuged at 21300 rcf for 10 min to remove thesupernatant, and the process is repeated two more times. The pellet isre-suspended in 1×TE buffer with 0.1% Triton X100 (1 ml). The reactionmixture is centrifuged at 21300 rcf for 10 min to remove thesupernatant, and the process is repeated one more time. The pellet isre-suspended in 1×TE buffer with 0.1% Triton X100, and the reactionmixture is centrifuged at 21300 rcf for 10 min to remove thesupernatant. The process is repeated one more time, and the pellet isre-suspended in 1×TE buffer with 0.1% Triton X100 (500 uL).

Example 3 Activation of an Amino-Hydrogel and Conjugation of theHydrogel with Amine Terminal DNA Probe

To a solution of 100 billion of amino-hydrogel (diameter=0.55 microns,23 million amines/micron3) in anhydrous, amine-free N-methylpyrrolidone(NMP) (600 μL) is added solid bis-succinimidyl suberate (22.1 mg, 60μmole), followed by tributylamine (14 μL, 60 μmole). After stirring at60 C for 1 h the hydrogels are isolated by centrifugation (30 min at21300 rcf). The hydrogel pellet is diluted with amine-free anhydrous NMP(1 ml) and isolated by centrifugation; this washing process is repeated2 times, and the final pellet is re-suspended in NMP (600 μL). Thishydrogel suspension is treated with acetic anhydride (30 μL, 317 μmole)and tributylamine (30 μL, 126 μmole), and is stirred at room temperaturefor 2 h. The resulting hydrogel is isolated by centrifugation (30 min at21300 rcf) and the pellet is diluted with amine-free anhydrous NMP (1ml) and isolated by centrifugation; this washing process is repeated 2times, and the final activated, capped pellet is diluted with 1 □μmoleof a 3 molar NMP solution of tetrabutylammonium5′-amino-oligonucleotide, tributylamine (1 □μmole), and amine-free NMPto a final volume of 600 μL. After stirring at 70 C for 16 h, the DNAconjugated hydrogel is isolated by centrifugation (30 min at 21300 rcf).The pellet is washed with NMP (1 ml), followed by Deionized water wash(1 ml) using centrifugation to isolate the pellets. The final hydrogelpellet is diluted with 1×TE buffer (1.6 ml) and stirred at 80 C for 1 h.The hydrogels are isolated by centrifugation (30 min at 21300 rcf) andwashed twice with DI water (1 ml) (using centrifugation for pelletisolation). To the final pellet is added 30% aqueous ammonia; after 15minutes at room temperature, the hydrogel is isolated centrifugation (20min at 21300 rcf) and washed 3× with DI water (1 mL) usingcentrifugation for isolation. The final pellet is re-dispersed in thebuffer desired for performing target amplification.

Example 4

Functionalization of a mesylated hydrogel with a mono-protected diaminewith subsequent deprotection, activation, and conjugation with anorganic soluble DNA probe is illustrated in FIG. 6.

To a pellet of 200 billion of mesylatedhydrogel (diameter=0.6 microns)(concentrated by centrifugation in anhydrous, amine-freeN-methylpyrrolidone (NMP)) is added 1 mL of mono-t-Boc protecteddiamine. O-(2-Aminoethyl)-O′[2-(Boc-amino)ethyl]hexaethylene glycol as aas a 50 mM solution. After agitation for 12 hr at 70 C, the hydrogel isisolated by centrifugation, washed once with de-ionized water, andde-protected at room temperature in 1 mL of 30% aqueous HCl; after 1 hr,the hydrogel is isolated by centrifugation, and washed 5 times with 5 mLportions de-ionized water. The hydrogel is dehydrated by centrifugationof the aqueous suspension, and dilution with 1 mL of anhydrous, aminefree NMP; this process is repeated 3 more times. After the finalcentrifugation, the pellet is activated with 0.6 mL of 125 mM of an NMPsolution of disuccinimidyl carbonate, to which was added 30 μL oftributylamine; after 2 hr at room temperature, the activated hydrogel isisolated by centrifugation, and the pellet is washed 5 times with 1 mLportions of anhydrous, amine free NMP. To the resulting pellet is added0.5 mL of a 3 μmolar NMP solution of tetrabutylammonium5′-amino-oligonucleotide and tributylamine (30 μL); after agitation for12 hr at 70° C., the conjugated hydrogel is concentrated bycentrifugation and washed 3 time with 1 mL portions of NMP. Theconjugated hydrogel pellet is diluted with 1×TE buffer (1.6 ml) andstirred at 80 C for 1 hr and subsequently isolated by centrifugation.After washing twice with de-ionized water, the final pellet wasre-dispersed in the buffer desired for performing target amplification.

In a first aspect, a method of conjugating a substrate includesexchanging a counter ion associated with a biomolecule with a lipophiliccounter ion to form a biomolecule complex, dispersing the biomoleculecomplex in a nonaqueous solvent, and coupling the biomolecule complex toa substrate in the presence of the nonaqueous solvent.

In an example of the first aspect, the biomolecule is a polynucleotide.

In another example of the first aspect and the above example, thelipophilic counter ion is a lipophilic ammonium ion, a lipophilicphosphonium ion, a lipophilic arsonium ion, a lipophilic sulfonium ion,or a combination thereof. For example, the lipophilic ammonium ion is atetraalkylammonium, a tetraarylammonium, mixed alkyl and aryl ammonium,or a combination thereof. In an example, the lipophilic ammonium ion isselected from the group consisting of tetramethylamonium,tetraethylamonium, tetrapropylamonium, tetrabutylamonium,tetrapentylamonium, tetrahexylamonium, tetraheptylamonium,tetraoctylamonium, alkyl and aryl mixtures thereof, and a combinationthereof. In a further example, the lipophilic phosphonium ion istetraphenylphosphonium. In an additional example, the lipophilicarsonium ion is a tetraalkylarsonium, a tetraarylarsonium, a mixed alkyland aryl arsonium ion, or a combination thereof. In another example, thelipophilic arsonium ion is tetraphenylarsonium. In an example, thelipophilic sulfonium ion is a trialkylsulfonium ion.

In a further example of the first aspect and the above examples, thenonaqueous solvent is non-reactive with coupling groups on the substrateand the biomolecule.

In an additional example of the first aspect and the above examples, thenonaqueous solvent is polar.

In another example of the first aspect and the above examples, thenonaqueous solvent is an amide, a urea, a carbonate, an ether, asulfoxide, a sulfone, a hindered alcohol, or a combination thereof. Forexample, the amide or urea is selected from a group consisting offormamide, N,N-dimethylformamide, acetamide, N,N-dimethylacetamide,hexamethylphosphoramide, pyrrolidone, N-methylpyrrolidone,N,N,N′,N′-tetramethylurea, N,N′-dimethyl-N,N′-trimethyleneurea, and acombination thereof. In another example, the carbonate is selected froma group consisting of dimethyl carbonate, propylene carbonate, and acombination thereof. In an additional example, the ether istetrahydrofuran. In a further example, the sulfoxide or sulfone isselected from a group consisting of dimethylsulfoxide, dimethylsulfone,and a combination thereof. In another example, the hindered alcoholincludes tert-butyl alcohol.

In a further example of the first aspect and the above examples, thesubstrate includes a polymeric particle.

In an additional example of the first aspect and the above examples, thepolymeric particle includes a coupling group reactive with a reactivegroup of the biomolecule.

In an example of the first aspect and the above examples, the couplinggroup includes one of a nucleophile or an electrophile and the reactivegroup includes the other of the nucleophile or the electrophile.

In a second aspect, a method of conjugating a polymeric particleincludes exchanging a cationic counter ion associated with apolynucleotide with a lipophilic cationic counter ion to form apolynucleotide complex, the polynucleotide including a nucleophilic oran electrophilic reactive group. The method further includes dispersingthe polynucleotide complex in a non-reactive, nonaqueous solvent andcoupling the polynucleotide to the polymeric particle by nucleophilic orelectrophilic substitution, the polymeric particle including anelectrophilic group or a nucleophilic group.

In a third aspect, a polymeric particle includes a polymer conjugated toa polynucleotide using the method of any one of above aspects orexamples.

In a fourth aspect, a method of sequencing includes amplifying a targetpolynucleotide in the presence of an oligonucleotide conjugatedpolymeric particle to form the polynucleotide conjugated polymericparticle. The oligonucleotide is at least partially complementary to thetarget polynucleotide. The oligonucleotide conjugated polymeric particleis formed by the method of any of the above aspects or examples. Themethod further includes applying the polynucleotide conjugated polymericparticle to a sequencing device, applying a primer to the polynucleotideconjugated polymeric particle, incorporating a nucleotide, and detectingthe incorporating.

In an example of the fourth aspect, detecting includes detecting achange in ion concentration associated with incorporation of anucleotide.

In a fifth aspect, a method of isolating a target polynucleotideincludes contacting a first solution including the target polynucleotidewith a probe conjugated substrate. The probe of the probe conjugatedsubstrate is at least partially complementary to the targetpolynucleotide. The probe conjugated substrate formed by the method ofany of the above aspects or examples. The method further includeswashing the probe conjugated substrate while the target polynucleotideis coupled to the probe and releasing the target polynucleotide in asecond solution.

In an additional aspect, a method of conjugating an oligonucleotide to apolymer includes treating a polymer comprising amine functionality witha bis-NHS ester or a disuccinimidyl carbonate to form a functionalizedpolymer and treating the functionalized polymer with an amine terminatedoligonucleotide to form a conjugated polymer including theoligonucleotide.

In an example of the additional aspect, the method further includesproviding an initial polymer including hydroxyl functionality, treatingthe initial polymer to substitute a halogen for the hydroxylfunctionality, and further treating the initial polymer with a protectedamine terminated polyether to form the polymer comprising the aminefunctionality.

In another example of the additional aspect and the above example, thepolymer is in the form of a polymer particle.

In a further example the oligonucleotide undergoes a treatment of themethods of the above aspects and examples thereof.

While the above describes conjugation to polymer particles, suchconjugation methods can be applied to similarly functionalized polymerin various forms, such as beads, substrates, sheets, rods, variouslyshaped polymer forms, or any combination thereof.

Note that not all of the activities described above in the generaldescription or the examples are required, that a portion of a specificactivity may not be required, and that one or more further activitiesmay be performed in addition to those described. Still further, theorder in which activities are listed are not necessarily the order inwhich they are performed.

In the foregoing specification, the concepts have been described withreference to specific embodiments. However, one of ordinary skill in theart appreciates that various modifications and changes can be madewithout departing from the scope of the invention as set forth in theclaims below. Accordingly, the specification and figures are to beregarded in an illustrative rather than a restrictive sense, and allsuch modifications are intended to be included within the scope ofinvention.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having” or any other variation thereof, areintended to cover a non-exclusive inclusion. For example, a process,method, article, or apparatus that comprises a list of features is notnecessarily limited only to those features but may include otherfeatures not expressly listed or inherent to such process, method,article, or apparatus. Further, unless expressly stated to the contrary,“or” refers to an inclusive-or and not to an exclusive-or. For example,a condition A or B is satisfied by any one of the following: A is true(or present) and B is false (or not present), A is false (or notpresent) and B is true (or present), and both A and B are true (orpresent).

Also, the use of “a” or “an” are employed to describe elements andcomponents described herein. This is done merely for convenience and togive a general sense of the scope of the invention. This descriptionshould be read to include one or at least one and the singular alsoincludes the plural unless it is obvious that it is meant otherwise.

Benefits, other advantages, and solutions to problems have beendescribed above with regard to specific embodiments. However, thebenefits, advantages, solutions to problems, and any feature(s) that maycause any benefit, advantage, or solution to occur or become morepronounced are not to be construed as a critical, required, or essentialfeature of any or all the claims.

After reading the specification, skilled artisans will appreciate thatcertain features are, for clarity, described herein in the context ofseparate embodiments, may also be provided in combination in a singleembodiment. Conversely, various features that are, for brevity,described in the context of a single embodiment, may also be providedseparately or in any subcombination. Further, references to valuesstated in ranges include each and every value within that range.

What is claimed is:
 1. A method of sequencing, the method comprising:amplifying a target polynucleotide in the presence of an oligonucleotideconjugated polymeric particle to form the polynucleotide conjugatedpolymeric particle, the oligonucleotide at least partially complementaryto the target polynucleotide, the oligonucleotide conjugated polymericparticle formed by: exchanging a counter ion associated with anoligonucleotide with a lipophilic counter ion to form a biomoleculecomplex; dispersing the biomolecule complex in a nonaqueous solvent; andcoupling the biomolecule complex to a polymeric particle in the presenceof the nonaqueous solvent to form the oligonucleotide conjugatedpolymeric particle; applying the polynucleotide conjugated polymericparticle to a sequencing device; applying a primer to the polynucleotideconjugated polymeric particle; incorporating a nucleotide; and detectingthe incorporating.
 2. The method of claim 1, wherein the lipophiliccounter ion is a lipophilic ammonium ion, a lipophilic phosphonium ion,a lipophilic arsonium ion, a lipophilic sulfonium ion, or a combinationthereof.
 3. The method of claim 2, wherein the lipophilic ammonium ionis a tetraalkylammonium, a tetraarylammonium, mixed alkyl and arylammonium, or a combination thereof.
 4. The method of claim 3, whereinthe lipophilic ammonium ion is selected from the group consisting oftetramethylamonium, tetraethylamonium, tetrapropylamonium,tetrabutylamonium, tetrapentylamonium, tetrahexylamonium,tetraheptylamonium, tetraoctylamonium, alkyl and aryl mixtures thereof,and a combination thereof.
 5. The method of claim 2, wherein thelipophilic phosphonium ion is tetraphenylphosphonium.
 6. The method ofclaim 2, wherein the lipophilic arsonium ion is a tetraalkylarsonium, atetraarylarsonium, a mixed alkyl and aryl arsonium ion, or a combinationthereof.
 7. The method of claim 6, wherein the lipophilic arsonium ionis tetraphenylarsonium.
 8. The method of claim 2, wherein the lipophilicsulfonium ion is a trialkylsulfonium ion.
 9. The method of claim 1,wherein the nonaqueous solvent is non-reactive with coupling groups onthe substrate and the biomolecule.
 10. The method of claim 1, whereinthe nonaqueous solvent is polar.
 11. The method of claim 1, wherein thenonaqueous solvent is an amide, a urea, a carbonate, an ether, asulfoxide, a sulfone, a hindered alcohol, or a combination thereof. 12.The method of claim 11, wherein the amide or urea is selected from agroup consisting of formamide, N,N-dimethylformamide, acetamide,N,N-dimethylacetamide, hexamethylphosphoramide, pyrrolidone,N-methylpyrrolidone, N,N,N′,N′-tetramethylurea,N,N′-dimethyl-N,N′-trimethyleneurea, and a combination thereof.
 13. Themethod of claim 11, wherein the carbonate is selected from a groupconsisting of dimethyl carbonate, propylene carbonate, and a combinationthereof.
 14. The method of claim 11, wherein the ether istetrahydrofuran.
 15. The method of claim 11, wherein the sulfoxide orsulfone is selected from a group consisting of dimethylsulfoxide,dimethylsulfone, and a combination thereof.
 16. The method of claim 11,wherein the hindered alcohol includes tert-butyl alcohol.
 17. The methodof claim 1, wherein the polymeric particle includes a coupling groupreactive with a reactive group comprising the biomolecule.
 18. Themethod of claim 19, wherein the coupling group includes one of anucleophile or an electrophile and the reactive partner includes theother of the nucleophile or the electrophile.